Shrimp thrombospondin (TSP): presence of O-β1,4 N-acetylglucosamine polymers and its function in TSP chain association in egg extracellular matrix

We characterized the existence of O-β(1,4)-GlcNAc polymers (β1,4GNP) that were anchored on the O-linked glycosylation sites of shrimp thrombospondin (pmTSP-II). There were five putative β1,4GNP linkages on the epithelial growth factor-like domain of pmTSP-II. Antibody against O-β-GlcNAc (CTD110.6) was used to prove the existence of linear and complex β1,4GNP. The antibody well reacted with linear chito-triose, -tetraose and -pentaose conjugated with phosphatidylethanolamine lipid. The immunoreactivity could also be detected with a complex β1,4GNP within pmTSP-II (at MW > 250 kDa). Upon denaturing the protein with SDS-PAGE buffer, the size of pmTSP-II was shifted to be 250 kDa, approximately 2.5 folds larger than the deduced molecular mass of pmTSP-II (110 kDa), suggesting additional association of pmTSP-II apart from its known disulfide bridging. This was confirmed by chitinase digestion on pmTSP-II protein leading to the subsequent smaller protein bands at 110–170 kDa in time- and concentration-dependent manners. These bands well reacted with CTD110.6 antibody and disappeared after extensive chitinase hydrolysis. Together, we believe that β1,4GNP on pmTSP-II serve the function in an inter-chain association to provide structural architecture of egg extracellular matrix, a novel function of pmTSP-II in reproductive biology.

www.nature.com/scientificreports/ is whether the N-linked or O-linked glycans (or both) on ZP3 participate in gamete interaction leading to acrosome induction [5][6][7] . Apart from being important receptors and inducers during fertilization processes, recently research has suggested that carbohydrate moieties play a non-fertilization role in regulating the structure of the mammalian egg ZP. It has been reported that all ZP glycoproteins interact both covalently and noncovalently among their filaments to form the helical-like bundle of extracellular matrix 8 . Nonetheless, detailed study of the types of carbohydrate species that are involved in the formation of this helical-like structure is still incomplete. Shrimp ovulated oocytes are surrounded by a jelly layer which is derived from the peripherally embedded cortical rod (CR) materials just prior to sperm interaction 9 . It has long been known that cortical rod components are heavily glycosylated, having about 70-75% carbohydrate constituents 10,11 . After egg spawning, the extruded CRs on the egg surface become a flocculent material known as egg water (EW) which is inherently an inducer of sperm acrosome reaction 12 . Among many CR proteins, thrombospondin protein (TSP) has been wellcharacterized in two separate shrimp species, termed TSP-I in F. chinensis [13][14][15] and pmTSP-II in Penaeus monodon 16,17 . As a multi-domain protein, it has been suggested that TSP has a broad range of physiological functions, including polyspermy barrier, anti-bacterial agent, ovarian development and inducer of acrosome reaction 13,14 .
Recently, the novel function of the N-linked mannosyl glycoconjugates on the calcium binding domain (TSP3 domain) of pmTSP-II has been shown to be crucial for sperm AR induction 17 . This finding suggests the role of carbohydrates in mediating the fertilization step of shrimp, in a similar manner to that reported in mammals, mentioned above. In this study, we extrapolated our findings to include O-linked glycoconjugates, β1,4GNP in the EGF-like domain of pmTSP-II and unraveled its role in TSP-interchain association, a novel non-fertilization role in shrimp gamete biology.

Results
The potential O-β-GlcNAc glycosylation sites on EGF-like domain of pmTSP-II. Our previous study reported that the full sequences of pmTSP-II comprised three important signature domains: chitin binding domain (CBD), EGF-like domain, and calcium-binding domain or TSP3 domains 16 . Here, we searched for the potential sites of O-β-GlcNAc glycosylation based on the CXXG(Y/F)(T/S)GZ 2-5 C amino acid sequence motif that was conserved throughout TSP's protein family 18 . This sequence could be found in many penaeid shrimp species including P. monodon, P. merguiensis, P. vannamei and P. japonicus (Fig. 1a). Interestingly, the O-β-GlcNAc glycosylation sites were found exclusively within the middle part of the EGF-like domain. In P. monodon, up to five O-β-GlcNAc anchoring sites were found. These numbers, however, varied from one (in P. merguiensis), to six (P. japonicus) and up to the maximum of eight potential sites in P. vannamei (red bold capital letters with gray shading). The site was, however, missing in P. chinensis, even though the TSP sequence in this species is closely related to other penaeid shrimp species 16 . In the two-dimensional linear arrangement of multidomains in pmTSP-II, the five positions of O-β-GlcNAc were evenly distributed within the EGF-like domain (Fig. 1a, blue box). The deduced three-dimensional structure of pmTSP-II suggested that the β1,4GNP exposed outward from the surface of pmTSP-II molecule (Fig. 1b). We thus believe that this arrangement of pmTSP-II would favor the interaction between β1,4GNP and CBD in the adjacent pmTSP-II peptide chain to form the supramolecular architecture of pmTSP-II.
Detection of O-β-GlcNAc residues on the conjugated lipid and the isolated CR proteins. As the deduced amino acid sequence of pmTSP-II indicated the anchorage of O-β-GlcNAc residues, we therefore aimed to investigate the existence of the complex O-β-GlcNAc chitin chain in the purified pmTSP-II using a CTD110.6 antibody. Since the antibody has been proven to recognize O-β-GlcNAc monosaccharide 19 , its binding with the oligomeric O-β-GlcNAc linked to lipid substrate was initially tested. The thin layer chromatograph in Fig. 2a clearly demonstrated a successful linkage of N-acetyl-triose, -tetraose and -pentaose (as a purplishblue enzyme precipitate resulting from the charring reaction of carbohydrates with an orcinol) to the PE lipid. The longer the sugars attached to the PE lipids, the slower the band mobility observed. Through an ELISA analysis, these oligomeric sugar-lipid linkages were well recognized by a CTD110.6 antibody with a dilution factor ranging from 1:12.5 to 1:50 and optical density from 0.05 to 0.3 (Fig. 2b). This result extrapolated the recognition of this antibody from monomeric sugar towards linear chained sugars.

Localization of O-β-GlcNAc modification on the cortical rods of mature shrimp ovaries. It
has been shown that the deduced molecular mass of a nascent peptide of pmTSP-II (without glycosylation) is approximately 110 kDa 16 . Upon its fresh purification by FPLC and dissolving by SDS-PAGE, the major band of 250 kDa was achieved (Fig. 3a, lane 1). However, after leaving the protein in suspension for a few weeks without protease inhibitors, the 250 kDa band was gradually hydrolyzed into the serially smaller bands at approximately 170, 150 and 110 kDa (Fig. 3a, lane 2). These proteins intensively reacted with anti-TSP (Fig. 3a, lane 4), suggesting that they are the multimeric forms existing in the extracellular matrix of the eggs. Using the CTD110.6 antibody to probe the purified pmTSP-II, similar immunoreactive bands as those of anti-TSP namely, 250 kDa, 170 kDa, 150 kDa and 110 kDa were revealed (Fig. 3a, lane 3, arrow heads). Interestingly, incubation of purified proteins with deglycosylation agents (trifluoromethanesulfonic (TFMSA) and sodium metaperidate (NaIO 4 ) resulted in a decreased intensity of the 250 kDa band and the appearance of 150 kDa and the other smaller banded proteins (Fig. 3b, lanes 2 and 3). In addition,exposure of the purified 250-kDa protein to strong denaturing agents did not grossly affect the intensity of the 250 kDa band, suggesting that the structural organization of the 250 kDa pmTSP-II is rather dependent on interaction of carbohydrate than disulfide bridges or hydrogen bonding.
We further performed the localization of β1,4GNP in vivo using paraffin sections of the mature shrimp ovary. Strong immunoreactivity of CTD110.6 antibody was detected on the peripheral rim of the CRs in the stage IV www.nature.com/scientificreports/ oocyte (Fig. 3c,d, arrowheads). A moderate staining could also be seen in the cytoplasmic granules of the stages II and III oocytes. Negative control revealed minimal antibody staining in the stage IV oocyte (Fig. 3e).

β1,4GNP of pmTSP-II is involved in the supramolecular chain-association. Despite many reports
indicating the presence of cysteine-based disulfide bridging that is involved in a supra-molecular structure of mammalian TSP proteins (whose deduced molecular mass is about 110-120 kDa) 20 , our native gel electrophoresis showed the clustered of pmTSP-II protein at the molecular mass much higher than 250 kDa (Fig. 4a, left panel, lane 2) which was intensely reactive with CTD110.6 antibody (right panel, arrowhead). Under the denaturing condition of SDS-PAGE, the results demonstrated a still-sizable 250 kDa pmTSP-II, even though they were exposed to a strong reducing agent and detergent in the PAGE resolving buffer (Fig. 4b, upper panel, lane PBS). We therefore aimed to search for the supramolecular cross-linkage of pmTSP-II strands, one of which could be a carbohydrate binding with its complimentary peptide domain, CBD. A bioinformatics analysis of pmTSP-II sequence, contained both an O-β-GlcNAc anchorage site on the N-terminus EGF-like domain as well as several CBD in the middle part of the molecule (Fig. 1a), a unique feature of many reported crustacean www.nature.com/scientificreports/ TSPs. Cross-interaction between β1,4GNP and CBD could thus, hypothetically, generate a multimeric-bundling structure of pmTSP-II. We tested this hypothesis by a chitinase digestion of the β1,4GNP on the freshly purified pmTSP-II (mainly 250 kDa) which should have dissolved its bundling structure, resulting in a singlet chain of a 110 kDa protein.
In the native condition, pmTSP-II that was treated with 0.25-1 U chitinase (24 h) showed the gradually reduced molecular mass of pmTSP-II to be the smaller sized proteins, particularly 110 and 150 kDa bands (arrowheads) and some other smaller bands (Fig. 4a, left panel, lanes 3-5). Reactivity of these digested proteins with a CTD110.6 antibody was also detected at 250 kDa band (in a decreased intensity manner upon increasing chitinase concentrations) and as a faint to moderate intensity at 150 and 110 kDa bands (Fig. 4a, right panel, lanes 2-4). In denaturing SDS-PAGE (Fig. 4b), it was apparent that the molecular mass of pmTSP-II gradually reduced to the smaller bands of 110-170 kDa bands (Fig. 4b, upper panel). Either longer exposure to 0.25 U chitinase (3-24 h) or higher amounts of enzyme (0.5-1 U) led to an apparent reduction of the upper bands (250 and 170 kDa) while increasing the intensity of the 150 and 110 kDa bands (lanes [12][13][14][15][16][17][18]. Moreover, when the purified pmTSP-II proteins were probed with a CTD110.6 antibody, the 250 kDa non-digested protein was intensely reactive with the antibody (Fig. 4b, lower panel, lanes 1-2). Reactivity of CTD110.6 antibody gradually shifted towards the lower molecular weighted bands, particularly the 110 kDa protein, in the 0.1-0.25 U chitinase-digested proteins (lanes 8-10). The prolonged or intensely digested samples with 0.5-1 U chitinase (lanes 12-18) showed very low or complete absence of antibody reactivity, suggesting a complete removal of β1,4GNP from the pmTSP-II peptide core. The results thus indicated the significance of β1,4GNP within the EGF-like domain in the supramolecular association of pmTSP-II within the egg extracellular matrix.

Discussion
The significance of carbohydrate moieties in egg vestments, either N-linked or O-linked glycoconjugates has become unraveled in the fertilization process, both in mammals and invertebrates 12,21,22 . However, there is less evidence demonstrating the structural role of specific carbohydrate moieties, even in model animals, such as mice and sea urchins. Among mouse ZP glycoproteins, some suggestions have been made about the involvement of carbohydrates in ZP structural formation 6,8 . Experimental evidence suggested that molecular bundling of ZP filaments can be dissolved in various conditions such as mild acid, heat and strong reducing agents, suggesting the involvement of both disulfide bridging and other non-covalent bonding such as carbohydrate-carbohydrate interaction 6,7,23 . If this is the case, the specific carbohydrate types involved in this interaction need to be explored. In marine invertebrates, we believe that structural organization of the egg vestments is more dependent on carbohydrate-carbohydrate or carbohydrate-protein interaction. This is simply due to their structural appearance which is more gelatinous 24  www.nature.com/scientificreports/ P. monodon, P. vannamei and P. japonicus contain 5-8 O-β-GlcNAc modification sites, whereas P. merguensis has only one site. It is noted in one penaeid shrimp species, P. chiensis, that even with the presence of EGF-like domain, but it still lacks an O-β-GlcNAc glycosylation site (Fig. 1a), an interesting evolutionary difference that remains to be addressed. Generally, protein polymerization to form the supramolecular structure through the disulfide bridges has been a priority. This also holds true in the case of mammalian TSPs that have previously been documented 27,28 . In such cases, deterioration of disulfide bridging with strong reducing agents greatly affects protein structure and produces TSP chain singlets 29 . Among shrimp TSPs studied, their analyzed molecular constituents also comprised many putative cysteines that potentially form the disulfide bonds needed for higher molecular hierarchical formation [13][14][15][16] . This is also the case of pmTSP-II where its deduced amino acid sequences are composed of all signature domains of TSPs as well as many putative cysteine residues. However, the fact that exposing pmTSP-II to the reducing agents did not affect a 250-kDa polymer of pmTSP-II, but rather treating it with chitinase enzyme would favor the chitin-based polymerization of pmTSP-II (Fig. 4). Therefore, the presence of several chitin binding domains (CBD) in shrimp TSPs would thus be an alternative bonding for clustering adjacent TSP chains to form bundles of TSP polymers. In fact, the role of β1,4GNP in molecular bundling has been well documented for the formation of many crustacean shells through a side-by-side stacking of chitins which are held together by hydrogen bonding 30,31 . This ends up with a super-large molecular mass of chitin polymers in several kinds of chitin-based shells or cuticles. Alternatively, the other bonding type of chitin chains with their complimentary sites on the nascent peptides (known as CBD) has also been mentioned in some literature 32 but not for shrimp TSPs. This latter bonding type of β1,4 GNP or chitin would be more favorable for pmTSP-II because of the existence of a number of CBD in the entire crustacean TSP molecule. Bundling of TSP chains through β1,4GNP-CBD interaction would also help to explain why shrimp TSPs engage many CBDs in their molecular structures which are rather unique to these animal species. www.nature.com/scientificreports/ Shrimp TSPs have been known to serve several functions, including cellular immune responses [33][34][35] or fighting against bacterial infection 13,14 . It is still unclear whether these physiological functions would have been derived from their peptide core or carbohydrate moieties. In the case of P. monodon, the significance of both N-and O-linked glycoconjugates in pmTSP-II was evident and summarized in Fig. 5. The N-linked mannose glycoconjugates in CBD and TSP3 domains have been shown to be involved in AR induction 17 . β1,4GNP presumably anchored in the EGF-like domain was characterized by its reactivity with CTD110.6 antibody (Figs. 3 and 4) and motif was also analyzed. The 3D structure was deduced from a known sequence of pmTSP-II using the Phyre2 server program (http:// www. sbg. bio. ic. ac. uk/ phyre2/ html/ page. cgi? id= index) 36 and analyzed by a PyMOL software (The PyMOL Molecular Graphics system, Schrodinger, LLC).

Isolation of cortical rods (CRs) and purification of pmTSP-II.
Wild-caught fully mature female P. monodon weighing 400-500 g captured from the Gulf of Thailand were obtained from a commercial farm in Chachoengsao province (Thailand). They were acclimatized in 500-L plastic laboratory tanks at the ambient temperature (28 °C) with seawater at the salinity of 20 ppt at least one day before they were used in the experiments. Shrimps were handled according to the guidelines of the Animal Care Committee, Mahidol University (MUSC-IACUC, protocol # 2016/014). Briefly, they were anesthetized by placing in ice for 5 min and dissected carefully to collect the ovaries. The pieces of mature ovary were fixed in Davidson fixative (Sigma-Aldrich, St. Louis, MO) and processes for immunohistochemistry or kept frozen for CR isolation. Isolation of CRs was performed according to previously described protocol 12 . Briefly, mature ovary was homogenized in an isolation medium (IM: 500 mM NaCl, 9 mM CaCl 2 , 14 mM KCl, 15 mM MgCl 2 , and 10 mM Tris, pH 7.6) containing 30% sucrose. Thereafter, the suspension was centrifuged at 1000 × g for 5 min to harvest the pellet, which was then resuspended in IM and overlaid in 40% sucrose, followed by centrifugation (8000 × g, 4 °C, 60 min). The yolk contaminants were washed away from CRs by single step centrifugation (1000 × g, 4 °C, 5 min) through a 30% sucrose in IM. The CR was then subjected to protein extraction in lysis buffer with and without protease inhibitor (1 mM PMSF + protease inhibitor cocktails (Sigma-Aldrich, St. Louis, MO). Protein concentration was determined by a BCA assay kit (Thermo Fisher Scientific, Waltham, MA). Purification of pmTSP-II protein was then performed as described 17 through Sepharose G-300 size-exclusion chromatography, (Amersham Pharmacia, Uppsala, Sweden) following the manufacturer's instructions. All chemicals were obtained by Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO).

Detection of naturally anchored O-β-GlcNAc polymers. Approximately 10 µg of purified pmTSP-
II proteins were dissolved in either native loading buffer or SDS-PAGE loading buffer. Each sample was then electrophoresed on 12.5% polyacrylamide under both non-reducing and reducing conditions. The gels were then stained by FASTsilver™ Gel staining kit (Merck, Darmstadt, Germany) or Coomassie blue staining (Merck, Darmstadt, Germany). For Western blotting, both native and reducing gels were electro-transferred onto a PVDF membrane (Merck-Millipore, Darmstadt, Germany). The membrane was treated with a blocking solution (1% BSA in PBS containing 0.1% tween) for 1 h at room temperature and then incubated with 0.4 μg/ ml (1:500) of a CTD110.6 monoclonal antibody overnight at 4 °C. After an extensive wash with PBST, the membrane was further incubated with 1:2,500 horse radish peroxidase (HRP) conjugated goat anti-mouse IgG (Abcam, Cambridge, UK) (room temperature, 2 h). The antigen-antibody complex was visualized by enhanced chemiluminescent method using ECL detection kit (Merck, Darmstadt, Germany). The resolved proteins on the PVDF membrane were exposed to the anti-TSP antibody 16 and the corresponding HRP-conjugated secondary antibody in the same conditions described above.

Immunolocalization of O-β-GlcNAc polymers in shrimp ovarian tissues.
Mature ovarian were fixed with Davidson's fixative overnight, then embedded in paraffin solution. The paraffin sections of mature ovary (5-μm thick) were dewaxed with xylene, rehydrated with a decreasing grading alcohol, then treated with 1% hydrogen peroxide (H 2 O 2 ) to quench any endogenous peroxidase activity. Subsequently, the sections were incubated with a blocking solution (2% BSA in PBS containing 0.4% Triton-X) for 30 min at room temperature, followed by 2 μg/ml (1:100) dilution of CTD110.6 antibody at 4 °C overnight. Thereafter, the sections were treated with 1:500 goat anti-mouse IgG conjugated with HRP (Abcam, Cambridge, UK) for 2 h at room temperature. The enzymatic product was visualized by a Nova Red peroxidase substrate kit (Vector laboratories, Burlingame, CA). Sections were counterstained with 0.1% hematoxylin and photographed using a DM3000 Leica microscopy.
Chitinase digestion and testing of the remaining glycoconjugates on pmTSP-II. The function of naturally conjugated O-β-GlcNAc in TSP chain association was tested by a chitinase digestion experiment. An equal volume of the purified pmTSP-II (1 mg/ml) was subjected to 1, 0.5, 0.25 and 0.1 units (U) chitinase digestion (Sigma Aldrich, specific activity is 200 U/mg) at the various time points (3 and 24 h) with a gentle agitation. The reactions were stopped by placing the mixtures on ice (4 °C). Alternatively, the purified pmTSP-II proteins were also treated with deglycosylation agents, trifluoromethanesulfonic (TFMSA) 37 and sodium metaperiodate (NaIO 4 ) (Sigma-Aldrich), as well as strong denaturizing agents, 7 M urea in 0.1 N HCl (Sigma-Aldrich) and 6 × loading buffer. The samples at various enzyme concentrations and digestion conditions were then resolved by gel electrophoresis either under a 5-20% continuous gradient native condition or SDS-PAGE, followed by silver staining. These digested pmTSP-II proteins were transferred onto PVDF membrane and probed with a CTD110.6 antibody to visualize the remaining O-β-GlcNAc residues on pmTSP-II protein under the antibody probing conditions described above.
Ethical approval. All animals procedures were conducted in accordance with the ethical standards of the guideline of the Animal Care Committee, Mahidol University (MUSC-IACUC, protocol no. 2016/014).

Data availability
The datasets analyzed during the current study are available in the National center for Biotechnology information (NCBI) (https:// www. ncbi. nlm. nih. gov) repository.